Development
Abstract
Renin cells are essential for survival and serve as key regulators of blood pressure and fluid-electrolyte homeostasis. Their function and identity are dependent on signals from their local microenvironment afforded by neighboring cells and nerves. Whether and how renin cells contribute to the development and maintenance of this microenvironment remains unclear. Because renin cells are rare -0.01 % of kidney cells- conventional histological approaches cannot capture their interaction with nerve fibers and surrounding cells within the nephron and its vasculature. Using high-resolution 3D imaging, cell-specific multicolor reporter mice, single-cell RNA-Seq, and conditional gene deletions, we mapped how renin cells assemble within arterioles and communicate with axon fibers to organize the growth and orientation of the kidney arterioles during development and disease. This co-inductive process is mediated by Ngf produced by renin cell precursors and is necessary for renin cell survival and innervation. Interestingly, renin enzymatic insufficiency elevates Ngf and drives arteriolar hypertrophy with aberrant axon sprouting and hyperinnervation. These findings indicate that renin cells regulate kidney neurovascular development revealing them as active organizers of their local neuroregulatory microenvironment in health and disease.
Authors
Manako Yamaguchi, Georgina Gyarmati, Liam McLaughlin, Hiroki Yamaguchi, Jason P. Smith, Lucas Ferreira de Almeida, Daisuke Matsuoka, Alexandre G. Martini, Sara M. Wilmsen, Sijie Hao, Kazuki Tainaka, Silvia Medrano, Sanjay Jain, Janos Peti-Peterdi, Maria Luisa S. Sequeira-Lopez, R. Ariel Gomez
Abstract
Airway structural remodeling and hyperresponsiveness (AHR), hallmarks of asthma, are influenced by genetic variations and adverse exposures. While intrauterine perturbations in lung development have been linked to adult pulmonary disease, the developmental origins of these abnormalities remain poorly understood. Here, we provide evidence of genetic background playing a key role in this process. Using A/J and C57BL/6J mice known for their distinct susceptibility to AHR, we show that A/J embryos selectively develop an aberrant airway smooth muscle (SM) program and AHR in adulthood when exposed transiently to a vitamin A/retinoic acid (RA)-disrupted intrauterine environment in vivo by maternal BMS493 administration. Single-nuclei multiomics identified a mesenchymal cell population overactivating TGFβ targets in response to BMS selectively in A/J lungs. These cells, localized to sites of airway SM initiation and pSMAD2-3, exhibited robust BMS-mediated upregulation of SMAD2-3 targets, including regulators of SM program Pdgfra and Tnc. Functional analyses in vivo and cultured lungs showed aberrant SM formation in areas of overactive TGFβ of BMS-exposed lungs. These abnormalities were prevented by inhibiting TGFβ signaling in utero in RA-deficient embryos. These findings underscore how distinct genetic backgrounds respond to intrauterine perturbations that program airway structure and function, with potential lasting consequences in postnatal pulmonary function.
Authors
Takehiro Otoshi, Benjamin D. Kotton, Ayyappa K.S. Kameshwar, Yoshinori Seki, Zachary Cardell, Xiangyi Ke, Yuta Matsuno, Pooja Rajaram, Youn-Kyung Kim, Sarah M. Sharpton, Loredana Quadro, Wellington V. Cardoso, Masako Suzuki
Abstract
The distal nephron segments play a critical role in maintaining electrolyte balance, yet the mechanisms that preserve epithelial identity and segmental organization within this region remain poorly defined. Yes-associated protein (YAP), a key effector of Hippo signaling, is essential for kidney development, but its function in distal nephron epithelia is unknown. Using a genetic gain-of-function approach to activate YAP selectively in distal nephron segments, we found that sustained YAP activity profoundly disrupts epithelial organization and nephron patterning. Lineage tracing revealed that both distal convoluted tubule and connecting tubule cells originate from Slc12a3-expressing cells, and YAP activation in these segments led to increased proliferation, displacement of lineage-labeled cells beyond expected segment boundaries, and loss of segment-specific gene expression. These changes were accompanied by defects in apicobasal polarity and junctional integrity, consistent with epithelial plasticity. Unexpectedly, YAP activation in distal nephron segments also suppressed proximal tubule gene expression, indicating non-cell-autonomous effects on nephron differentiation. Together, these findings identify YAP as a critical regulator of epithelial identity in the distal nephron segments and reveal a previously unrecognized role for Hippo signaling in coordinating intersegmental organization during kidney development.
Authors
Zeinab Dehghani-Ghobadi, Eunah Chung, Mohammed Sayed, Christopher Ahn, Hyojin Alex Choi, Annissa Aamoum, Benjamin R. Thomson, Yueh-Chiang Hu, Hee-Woong Lim, Joo-Seop Park
Abstract
Several genes guide inner ear development, and mutations in these genes can cause malformations that result in congenital hearing loss. However, the contribution of noncoding regulatory elements remains largely unclear. This study investigates the function of distal enhancer elements in the transcriptional regulation of GDF6, a gene implicated in cochlear development. Using mouse models with targeted deletions, human inner ear organoids, and CRISPR interference (CRISPRi), we identified a downstream regulatory interval harboring a developmental enhancer required to maintain GDF6 expression during otic epithelial maturation and cochlear morphogenesis. Deletion of this regulatory region or targeting of CRISPRi-based repressors to these regions resulted in decreased GDF6 expression, failure of otic-epithelium development, and prevention of hair cell-like differentiation, reflecting cochlear aplasia observed in patients with corresponding genomic deletions. These findings highlight the contribution of long-range regulatory elements to auditory development and illustrate how their disruption contributes to human deafness.
Authors
Mohammad Faraz Zafeer, Clemer Abad, Havva Ortabozkoyun, Memoona Ramzan, Guney Bademci, Maria C. Robayo, Duygu Duman, Rolen M. Quadros, Shengru Guo, Juan I. Young, Anthony J. Griswold, Channabasavaiah B. Gurumurthy, Derek M. Dykxhoorn, Katherina Walz, Mustafa Tekin
Abstract
While Wilms tumors commonly arise from renal precursor cells and maintain features of the developing kidney, recent studies have demonstrated significant genetic, histologic, and molecular heterogeneity. To further investigate tumor variability as well as unifying features in tumor biology, we performed single nuclei RNA-sequencing (snRNA-seq) on treatment naïve, favorable histology Wilms tumors utilizing a reference atlas established from tumor-adjacent kidney samples and fetal kidney. Transcriptional profiles of blastemal, stromal, and epithelial components were correlated with tumor histology and demonstrate developmental-lineage plasticity, with PAX2 and PAX8 expression normally restricted to the nephron lineage of the fetal kidney found to be expressed in tumor stroma, as well as the stromal marker POSTN identified in tumor blastema. Further analyses of the blastema show shared transcriptional features with the differentiation trajectory of “uninduced” to “early differentiating” fetal nephron progenitor cells as well as aberrant expression of stromal signatures. A number of pathways from fetal nephron progenitors were maintained in the blastema, including regulation of stem cell maintainence and axonogenesis, whereas other pathways appear enriched in specific tumor samples, demonstrating the ability of snRNA-seq to identify both unifiying transcriptional signatures and uncover distinct molecular targets in signaling pathways and/or biological drivers of Wilms tumorigenesis.
Authors
Mike Adam, Keri A. Drake, Naomi Pode-Shakked, Katherine VandenHeuvel, Steve Potter, James Geller
Abstract
Small bowel transplantation (SBT) is the only curative treatment for intestinal failure due to short bowel syndrome (SBS); however, the 10-year graft survival rate after SBT remains below 50%. Therefore, alternative treatments are required. We developed a new therapeutic strategy for intestinal failure involving in vivo intestinal regeneration using a decellularized scaffold in a rat model. A 3-cm segment of decellularized small intestine was anastomosed to the jejunum for in vivo regeneration. After four weeks of regeneration, the entire native intestine was resected to induce SBS, and the regenerated intestine was transplanted into the same rat. Histological analysis revealed regeneration of mucosa, nerves, muscular layer, and crypts, consistent with autologous cell infiltration. An indocyanine green test confirmed blood flow from the adjacent mesentery into the regenerated intestine. The regenerated intestine exhibited absorption of nutrients in vivo, and ex vivo assessments confirmed peristalsis and absorptive capacity comparable to native intestine. Transplantation of the regenerated intestine significantly improved postoperative nutritional status in SBS rats. Our method, autogenic-regenerated intestinal transplantation, showed the therapeutic potential for intestinal failure. This is the first study to demonstrate a functionally integrated regenerated intestine, providing a foundation for future regenerative therapy.
Authors
Kentaro Iwaki, Takamichi Ishii, Hidenobu Kojima, Fumiaki Munekage, Hiroshi Horie, Kenta Makino, Takuma Karasuyama, Yusuke Hanabata, Elena Yukie Uebayashi, Satoshi Ogiso, Etsuro Hatano
Abstract
Fibroblasts in the lung mesenchyme produce growth factors and extracellular matrix components that guide formation of distal airspaces during the saccular stage of lung development. Inflammation in preterm infants disrupts this process, leading to bronchopulmonary dysplasia (BPD). To examine how mesenchymal inflammation contributes to BPD pathogenesis, we developed a transgenic mouse model (“IKKβTbx4”) in which expression of activated human IκB kinase beta (IKKβ), an upstream activator of NF-κB, was induced in Tbx4 lung enhancer-positive mesenchymal cells during the saccular stage of lung development (postnatal day 0 [PN0] - PN5). Saccular stage IKKβTbx4 mice exhibited a BPD-like phenotype with interstitial thickening and reduced distal airspaces at PN5, progressing to emphysematous enlargement of the distal lung at 2 mo of age. Mesenchymal NF-κB activity upregulated the chemokines CCL2 and CCL7, recruiting CCR2pos monocyte-derived macrophages to the lung. Recruited macrophages disrupted the elastin scaffold and impaired microvascular organization with reductions in CAP2 endothelial cells (aCaps) and pericytes. Blocking CCR2-dependent monocyte recruitment with a small molecule CCR2 antagonist rescued the abnormal lung phenotype. These findings identify mesenchyme-macrophage crosstalk as a mechanism by which inflammation disrupts saccular stage lung development, suggesting a role for this signaling axis in BPD pathogenesis.
Authors
Benjamin C. Crawford, Jessica Chauviere Lee, Bertha C. Elias, Shivangi Dave, Riet van der Meer, Wei Han, Alexandria L. Sharkey, David S. Nichols, Charles Shissias, Lauren Pate, Hayden Tan, Dawn C. Newcomb, Wei Shi, Lawrence S. Prince, Erin J. Plosa, Bradley W. Richmond, Timothy S. Blackwell, Susan H. Guttentag, John T. Benjamin
Abstract
Although inflammatory complications are common in preterm infants, the effects of these conditions on neonatal immune development remain poorly defined. We therefore investigated whether severe bronchopulmonary dysplasia (BPD) and systemic infection, two major complications of prematurity, produce distinct immune signatures and change immune composition over time. We performed longitudinal high-dimensional immune profiling of residual whole blood from 38 preterm infants sampled every two weeks, along with 10 term infants at birth. Preterm infants with severe BPD showed a progressive increase in Th17-polarized CD4+ T cells, neutrophils, and Th17-related cytokines compared to age-matched infants with moderate BPD. In contrast, some preterm infants with systemic bacterial or viral infections mounted exceptionally robust CD8+, CD4+, and γδ T cell responses, with oligoclonal expansion, terminal differentiation, and coordinated plasma cytokine shifts that persisted well beyond resolution of infection. These findings demonstrate that different preterm comorbidities imprint the neonatal immune system in divergent ways. Thus, comprehensive and longitudinal immune profiling may not only identify connections between clinical inflammatory complications and underlying immune pathways but also reveal potential targets for intervention.
Authors
Benjamin A. Fensterheim, Michelle L. McKeague, Divij Mathew, Shwetank, Ajinkya Pattekar, Matthew Lee, Zahabia Rangwala, Sean Nasta, Macy C. Kee, Cynthia Clendenin, Zachary Martinez, Caroline Diorio, Allison R. Greenplate, Krithika Lingappan, E. John Wherry
Abstract
X-linked myotubular myopathy (XLMTM) is a rare genetic disorder that typically presents at birth with progressive muscle weakness and respiratory difficulties and is caused by myotubularin-1 (MTM1) gene mutations. Here we examine the role of phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta (PIK3C2B), a lipid kinase that interacts with MTM1, in XLMTM in various models. We examined the effect of BLU3797, a novel, highly potent, selective, orally bioavailable PIK3C2B inhibitor, on survival, muscle development, myofiber phenotypes, and gene expression in MTM1-/y mice. PIK3C2B-deficient XLMTM animals demonstrated increased survival, restored muscle function, fewer myofibers with centralized nuclei, and normalization of disease-associated molecular markers. BLU3797 alleviated the XLMTM phenotype in a dose-dependent and reversible manner. Loss of functional PIK3C2B in XLMTM mice promoted a more differentiated, adult-like myofiber profile, which was strongly associated with normalization of disease surrogates and a reduction in markers of early muscle development and regeneration. BLU3797 treatment appears to modulate the expression of microRNAs associated with satellite cell activation and myofiber fusion. These findings indicate that PIK3C2B inhibition with BLU3797 effectively reverses the XLMTM disease phenotype by enhancing muscle function and promoting development toward a more mature state.
Authors
Andrew Shearer, Melissa L. Brooks, Maxine M. Chen, Thiwanka Samarakoon, John Hsieh, Gramoz Kondakci, Emanuele Perola, Jason Brubaker, Kristina Fetalvero, Stefanie Schalm, Joana Caetano-Lopes
Abstract
β-arrestins are ubiquitously expressed cytosolic adaptor proteins that regulate G protein-coupled receptor-dependent and -independent pathways essential for numerous physiological functions. This study investigated the role of β-arrestin1 and -2 in embryonic lymphatic vessel development and survival by generating and characterizing mice with lymphatic, tamoxifen-inducible loss of the genes encoding β-arrestin-1 and -2 (Arrb1/2ΔiLEC). At embryonic day15.5 (E15.5), Arrb1/2ΔiLEC embryos exhibit profound hydrops fetalis and increased embryonic mortality compared to control Arrb1/2fl/fl embryos. Edematous Arrb1/2ΔiLEC embryos, which were more often represented by the female sex, showed growth restriction and decreased lymphatic endothelial cell (LEC) proliferation in the jugular lymphatic sac compared to controls. In vitro knockdown of β-arrestin1 in LECs increased proliferation and increased activation of AKT, while knockdown of β-arrestin2 decreased proliferation and decreased activation of both ERK and CREB. Arrb1/2ΔiLEC embryos also exhibited dilated dermal lymphatics with decreased continuous VE-Cadherin adherens junctions compared to controls. These results were recapitulated in vitro in β-arrestin1 and/or -2 knockdown human LECs, which showed a decrease in membrane VE-Cadherin and β-catenin levels, and prevention of adrenomedullin-induced linearization of VE-cadherin at endothelial cell–cell junctions. Collectively, these results demonstrate that loss of β-arrestin1/2 in lymphatics causes hydrops fetalis, mid-gestational growth arrest and embryonic demise associated with reduced LEC proliferation and disrupted VE-Cadherin adherens junctions.
Authors
Yanna Tian, D. Stephen Serafin, Monserrat Avila-Zozaya, Alyssa M. Tauro, Natalie M. Torres-Valle, Bryan M. Kistner, Danielle M. Dy, Elizabeth S. Douglas, Kathleen M. Caron
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